Delivery of biopharmaceuticals to local or systemic target sites via administration to mucosal surfaces is considered advantageous for several reasons, and is continuously explored by using optimized therapeutic molecules, novel excipients, and innovative formulation designs. However, the transport of biopharmaceuticals into and across epithelia in, for example, the airways and the intestine are limited due to their inherent instability and large molecular size. Thus, detailed knowledge about the properties of the epithelial barrier and the transport pathways that may be explored for delivery of peptides, proteins, and nucleotide-based drugs is a prerequisite for proper interpretation of the results of any experimental approach aiming at mucosal delivery of biopharmaceuticals. The present chapter describes the transport pathways and mechanisms relevant for epithelia in mucosal tissue providing examples of the drug delivery approaches taken to deliver biopharmaceuticals via the paracellular and/or transcellular pathways. Also, methodologies used to monitor and assess the delivery are critically discussed.